PROJECT SUMMARY Rotavirus (RV) is the leading cause of life-threatening diarrheal disease in children under the age of 5, and results in nearly half a million deaths annually, despite the use of two licensed RV vaccines. Although RV vaccines are available, they do not work optimally in countries where they are needed most. Human rotaviruses (HRVs) grow poorly in cultured cells and do not infect mice, which are only susceptible to animal rotavirus infection. We do not understand how HRV infections cause life-threatening disease and question if knowledge from existing animal models is fully applicable to HRVs. We pioneered establishing human intestinal enteroids (HIEs) as novel ex vivo model ?mini-gut? systems to examine host-pathogen interactions and pathophysiology. HIEs are self-organizing, 3-dimensional physiologically active, multicellular cultures that mimic the ordered structure of the intestine. We have found that HRV strains replicate extremely well in HIEs mimicking the host permissiveness observed in people, thus establishing a new model to study HRV infections and pathogenesis. We propose studies to directly examine mechanisms of pathophysiology and genetic control of HRV infection and growth. The recent discovery that cellular receptors used by HRV strains are histoblood group antigens (HBGAs) resulted in a paradigm shift in thinking about the host's genetic influence on RV infection and provides a new perspective on zoonotic transmission of rotaviruses. Unique differences in HBGA expression may explain the higher prevalence of unusual HRV strains in Africa and lower vaccine efficacy seen in distinct ethnic populations. We hypothesize that HRV infection of enteroids is genetically restricted and permissive infection leads to novel intracellular and extracellular innate signaling and response pathways that underlie HRV pathogenesis and pathophysiology. In addition, the outcome of HRV infection or vaccination can be modulated by beneficial microbes. We propose studies in Aim 1 to determine whether genetically-controlled modulators of susceptibility (HBGAs) and distinct innate responses in HIEs regulate HRV infection and pathophysiology (with Cores B and C). Studies in Aim 2 will establish the effect of commensal or pathogenic organisms on HRV or vaccine infection. These studies will elucidate the molecular basis for strain- specific host range restriction observed in children of different ethnic groups and be relevant to understanding vaccine properties. Furthermore, evaluation of modified HIE cultures that house human commensals, probiotics or other pathogens (with Cores B, C and Project 2) in new tailored biomaterial platforms (with Project 3) will lead to a better understanding of how epithelial responses to a viral infection are modulated. Collectively, this work will provide a new understanding of rotavirus pathophysiology and the subsequent critical adaptive responses in humans that trigger immunity and disease.